Effects of the root of Platycodon grandiflorum on airway mucin hypersecretion in vivo and platycodin D3 and deapi-platycodin on production and secretion of airway mucin in vitro

We investigated whether aqueous extract of the root of Platycodon grandiflorum A. de Candolle (APG), platycodinD₃ and deapi-platycodin significantly affect the production and secretion of airway mucin using in vivo and in vitro experimental models. Effect of APG was checked on hypersecretion of pulmonary mucin in sulfur dioxide-induced bronchitis in rats. Confluent NCI-H292 cells were pretreated with platycodinD₃ or deapi-platycodin for 30 min and then stimulated with PMA (phorbol 12-myristate 13-acetate) for 24 h. The MUC5AC mucin production and secretion were measured by ELISA. The results were as follows: (1) APG stimulated the secretion of airway mucin in sulfur dioxide-induced bronchitis rat model; (2) platycodinD₃ and deapi-platycodin inhibited the production of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively; (3) however, platycodinD₃ and deapi-platycodin did not inhibit but stimulated the secretion of MUC5AC mucin induced by PMA from NCI-H292 cells, respectively. This result suggests that aqueous extract of P. grandiflorum A. de Candolle and the two natural products derived from it, platycodinD₃ and deapi-platycodin, can regulate the production and secretion of airway mucin and, at least in part, explains the traditional use of aqueous extract of P. grandiflorum A. de Candolle as expectorants in diverse inflammatory pulmonary diseases.     

Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor

BackgroundThe root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine.Hypothesis/purposeWe investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.Study designConfluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA.Results(1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10⁻⁵ M (p < 0.05) and 10⁻⁶ M (p < 0.05) or PMA at 10⁻⁴ M (p < 0.05), 10⁻⁵ M (p < 0.05) and 10⁻⁶ M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10⁻⁴ M (p < 0.05) or PMA at 10⁻⁴ M (p < 0.05).ConclusionThese results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Influences of flavomycin,ropadiar, and saponin on nutrient digestibility,rumen fermentation,and methane emission from sheep

This study focused on the effects of three additives given together with a hay/concentrate-based diet on nutrient digestibility, rumen fermentation, and methane emission from sheep. The basal diet consisted of 1.29 kg mixed hay and 0.43 kg concentrate mixture based on dry matter (DM). Treatments consisted of control (no additive), flavomycin⁴⁰ (250 mg/d), ropadiar from an oregano extract (250 mg/d), and saponin in the form of a yucca schidigera extract (170 mg/d). Results indicated that intake and digestibility were unaffected by treatments (P>0.05). The NH₃-N concentration of rumen liquor was lower (P<0.05) for additive treatments versus the control treatment. Higher concentrations of volatile fatty acid (VFA) were observed in the saponin (75.8 mmol/L) and ropadiar (73.1 mmol/L) treatments. The proportion of individual fatty acid of rumen liquor was unchanged, whereas lower ratio of acetate to propionate in the saponin treatment was observed (P<0.05). The average methane production expressed on digested organic matter (OM) and neutral detergent fiber (aNDFom) basis were decreased by approximately 3.3 and 12.0 g/kg, respectively in saponin, and 4.2 and 11.9 g/kg in ropadiar treatment compared to the control. Methane production was positively correlated with the concentrations of NH₃-N, and negatively correlated with total VFA and the proportion of propionate of rumen liquor (P<0.05). The study found that saponin and ropadiar could have the potential to reduce rumen methanogenesis in sheep.     

Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees

The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥98%) after incubation for 24 h at 37 °C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 °C for 10 min or autoclaving at 121 °C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6 h and ≥95% degradation was observed after 24 h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography–mass spectrometry (LC–MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity.     

Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization

Sequential optimization strategy based on statistical experimental designs was employed to enhance glucan production by Leuconostoc dextranicum NRRL B-1146 in flask culture. A two-level Plackett–Burman design was employed first where 11 variables were studied for their influence on glucan production. Sucrose, peptone and yeast extract were the most significant variables improving glucan production. A three-level Box–Behnken factorial design was employed for maximizing the glucan production. A mathematical model was developed to show the effects of each medium component and their combinatorial interactions on glucan production. The optimal medium composition for maximum glucan production was sucrose 5.95%, peptone 0.52% and yeast extract 2.9%. This composition predicted 1063 mg/l glucan, the experimentally found glucan was 1015 ± 4.5 mg/l that showed a good agreement with the predicted value. The purified glucan was homogenous and its structural characteristics investigated by FT-IR, ¹H NMR and ¹³C NMR spectroscopic techniques showed that it contained α-(1 → 6) and α-(1 → 4) linkages.     

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (?log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.     

Anti-inflammatory secoiridoid glycosides from Gentianella azurea

A phytochemical investigation on crude extract of Gentianella azurea led to the isolation of ten new (1–10) and one known (11) secoiridoid glycosides. Their structures were unambiguously elucidated by analysis of 1D and 2D NMR. Compounds 2, 5 and 11 were found to inhibit nitric oxide (NO) production in RAW 264.7 macrophages with IC₅₀ values of 52.78 ± 8.61, 0.69 ± 0.23 and 5.18 ± 1.33, respectively, while indomethacin, the positive control, showed an IC₅₀ value of 1.25 ± 0.52 μM.     

Chemical properties and antiinflammatory activity of a galactomannoglucan from Arecastrum romanzoffianum

A galactomannoglucan (GMG) with an estimated weight-average molar mass of 1.5 × 10⁵ was obtained from an aqueous extract of the mesocarp of fruits of Arecastrum romanzoffianum (Cham.) Becc. by fractionation by Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that GMG has a chain of (1 → 4)-linked β-d-mannopyranosyl residues, a chain of (1 → 3)-linked β-d-galactopyranosyl residues, a chain of (1 → 4)-linked α-d-glucopyranosyl residues, repeating units of β-d-galactopyranosyl-(1 → 4)-β-d-mannopyranosyl and β-d-mannopyranosyl-(1 → 4)-α-d-glucopyranosyl and terminal residues of d-galactopyranosyl and d-glucopyranosyl which comprised galactose, mannose and glucose in the molar ratio of 10:37:53. The polysaccharide exhibited significant antiinflammatory activity against carrageenan-induced mouse paw oedema.     

Structural characterization of a pectic polysaccharide from Nerium indicum flowers

A polysaccharide fraction, J6, was isolated from the hot-water extract of flowers of oleander Nerium indicum Mill., using ethanol precipitation, cetyltrimethylammonium bromide (CTAB) complexing, anion-exchange chromatography and gel permeation chromatography. J6 was found to contain l-rhamnose, l-arabinose, d-galactose, and d-galacturonic acid, in the ratio of 10.1:49.8:30.1:10.0. Its structure was investigated by methylation analysis, periodate oxidation, Smith degradation, partial acid hydrolysis, electrospray ionization mass spectrometry and NMR spectroscopic methods. It was found that J6 is an RG-I type polysaccharide, which contains a rhamnogalacturonan backbone, with various branches attached to O-4 of l-rhamnose. The branches probably involve (1 → 4)-β-d-galactan, branched l-arabino-(1 → 3)(1 → 6)-β-d-galactan, and (1 → 5)-α-l-arabinan. J6 stimulated NO production of macrophage RAW264.7 cells in a preliminary test.     

Chemical properties and adjuvant activity of a galactoglucomannan from Acrocomia aculeata

A galactoglucomannan (GGM) with an estimated weight-average molar mass of 3.5 × 10⁵ was obtained from an aqueous extract of the mesocarp of fruits of Acrocomia aculeata (Jacq.) Lodd. by fractionation by Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that GGM has a main chain of (1 → 4)-linked β-d-mannopyranosyl residues attached to an initial chain of (1 → 3)-linked β-d-galactopyranosyl residues and a terminal chain of (1 → 4)-linked α-d-glucopyranosyl residues which comprised galactose, glucose and mannose in the molar ratio of 18:22:60. The adjuvant potential of the polysaccharide on the cellular immune response against ovalbumin antigen was investigated using in vivo assays.     

Directional secretion of prostaglandin F(2alpha) by polarized luminal epithelial cells from pig endometrium

In swine, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin. The capacity of luminal epithelial cells isolated from the endometrium of day 16 cyclic pigs, to secrete PGF(2alpha)500 Omega/cm(2)), they were treated on the apical, basal or both surfaces with 0 or 100 nM oxytocin (OT) in Experiment 1 or phorbol 12-myristate 13-acetate (PMA) in Experiment 2. In the absence of OT or PMA, PGF(2alpha) secretion occurred primarily from the basal surface and was approximately 12-fold greater (P < 0.001) than from the apical surface. Treatment with OT did not stimulate PGF(2alpha) secretion from either surface regardless of which surface was treated. In contrast, PMA increased PGF(2alpha) secretion from both surfaces. Treatment of the apical surface or both surfaces with PMA increased (P < 0.001) PGF(2alpha) secretion similarly from both surfaces. Treatment of only the basal surface with PMA increased (P < 0.01) PGF(2alpha) secretion from both surfaces, but tended (P = 0. 06) to increase its secretion from the basal surface more than from the apical surface. These results indicated that PGF(2alpha) secretion by luminal epithelial cells obtained from cyclic pigs occurs primarily toward a basal direction and is not stimulated by oxytocin. Activation of protein kinase C stimulates directional secretion of PGF(2alpha) from both surfaces of the epithelial cells.     

RANKL/OPG in primary cultures of osteoblasts from post-menopausal women. Differences between osteoporotic hip fractures and osteoarthritis

The OPG/RANKL/RANK system is important in the balance between bone formation and resorption.We used primary human osteoblasts (hOBs) cells to examine the impact of 17-β-estradiol (E2) or/and 1,25-dihydroxyvitamin D (1,25D) in OPG/RANKL system in 28 post-menopausal (PM) women; (a) with hip fracture (OP) or (b) with osteoarthritis (OA). The hOB from OP patients proliferated slower during the first stage, than the OA women (31.5 ± 2.6 and 21.4 ± 1.3 days, respectively, p < 0.05). The OP group secreted significantly higher OPG protein levels than the OA women (10.1 ± 2.6 and 4.4 ± 0.8 pmol/L, respectively, p < 0.05). The 1,25D and 1,25D+E2 induce an increase in RANKL and RANKL/OPG mRNA expression in OP patients above 200% (p < 0.05).HOBs from the osteoporotic hip initially proliferate slower but after reaching the first cellular confluence, the proliferation rate is equal in both groups. Furthermore, hOBs from hips with OP present a higher protein secretion of OPG, and higher RANKL and RANKL/OPG expression ratio in response to 1,25D and 1,25D+E2, than hOBs from OA women. All this could suggest that the greater bone loss that characterizes OP patients can be mediated due to differences in the secretion and expression of the RANKL/OPG system in response to different stimuli.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells

Cytokines are known to play pivotal roles in cancer initiation, progression and pathogenesis. Accumulating evidences suggest differences in basal and stress-induced cytokine profiles of cancers with diverse origin. However, a comprehensive investigation characterising the cytokine profile of various tumor types after acute and fractionated doses of gamma-irradiation, and its effect on survival of bystander cells is not well known in literature. In the present study, we have evaluated the cytokine secretion profile of human tumor cell lines (HT1080, U373MG, HT29, A549 and MCF-7) either before (basal) or after acute (2, 6 Gy) and fractionated doses (3 × 2 Gy) of gamma-irradiation in culture medium obtained from these cells by multiplex bead array/ELISA. Moreover, clonogenic assays were performed to evaluate the effect of conditioned medium (CM) on the survival and growth of respective cells. Based on the screening of 28 analytes, our results showed that the basal profiles of these cell lines varied considerably in terms of the number and magnitude of secreted factors, which was minimum in MCF-7. Interestingly, TNF-α, IL-1β, PDGF-AA, TGF-β1, fractalkine, IL-8, VEGF and GCSF were found in CM of all the cell lines. However, secretion of certain cytokines was cell line-specific. Moreover, CM caused increase in clonogenic survival of respective tumor cells (in the order HT1080 > U373MG > HT29 > A549 > MCF-7), which was correlated with the levels of IL-1β, IL-6, IL-8, GMCSF and VEGF in their CM. After irradiation, the levels of most of the cytokines increased markedly in a dose dependent manner. The fold change in cytokine levels was lower in irradiated conditioned medium (ICM) of tumor cells collected after fractionated than respective acute dose, except in MCF-7. Interestingly, amongst these cell lines, the radiation-induced fold increase in cytokine levels was maximum in ICM of A549 cells. Moreover, bystander A549 cells treated with respective ICM showed dose dependent decrease in clonogenic survival. In conclusion, present study revealed the similarities and subtle differences in basal and radiation-induced cytokine profile of different tumor cell lines, and its influence on growth and survival of respective bystander cells. These findings may add a new dimension to our current understanding about role of cytokines in cancer biology.     

An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae)

Norepinephrine-stimulated skin secretions of the Tyrrhenian painted frog Discoglossus sardus Tschudi, 1837 (Alytidae) did not contain any peptide with antimicrobial or hemolytic activity. However, peptidomic analysis of the secretions revealed the presence of an abundant peptide with structural similarity to frenatin 2, previously isolated from the Australian frog Litoria infrafrenata (Hylidae). The primary structure of the peptide, termed frenatin 2D, was established as DLLGTLGNLPLPFI.NH₂ by automated Edman degradation and mass spectrometry with electron-transfer dissociation (ETD)-based fragmentation and confirmed by chemical synthesis. The structure of a second frenatin 2-related peptide, termed frenatin 2.1D, that was present in much lower abundance was established as GTLGNLPAPFPG. Frenatin 2D (20 μg/ml) significantly stimulated production of the proinflammatory cytokines TNF-α (P < 0.05) and IL-1β (P < 0.01) by mouse peritoneal macrophages but the peptide did not potentiate the stimulation produced by lipopolysaccharide (LPS). The peptide increased IL-12 production in both unstimulated (P < 0.01) and LPS-stimulated (P < 0.05) cells but stimulatory effects on IL-6 production were not significant. The biological role of frenatin 2D is unknown but it is speculated that the peptide acts on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.     

Effects of Canarium odontophyllum leaves on plasma glucose and T lymphocyte population in streptozotocin-induced diabetic rats

Type 1 diabetes mellitus is a chronic disease characterized by lack of insulin production. Immune mechanisms are implicated in the pathogenesis of Type 1 diabetes. Canarium odontophyllum (CO) fruits and leaves have been shown to possess high antioxidant activity. This study was conducted to evaluate the effects of CO leaves aqueous extract on the blood glucose and T lymphocyte population in the spleen of streptozotocin (STZ)-induced diabetic rats. Nineteen male Sprague–Dawley rats were randomly divided into three groups: normal, diabetic control and CO treated diabetic groups. Diabetes was induced by a single intraperitoneal injection of 65 mg STZ/kg body weight. The extract of CO leaves was administered orally by force feeding daily at the dose of 300 mg/kg for 28 days. The rats were sacrificed at the end of the study and the spleen was harvested for flow cytometry analysis. The results showed a significant decrease in body weight of diabetic and CO treated diabetic groups compared with the normal group (p < 0.05). The fasting blood glucose level of CO treated diabetic group was significantly lower than the diabetic group (p < 0.05). Diabetic and CO treated diabetic groups showed a significant increase in the percentage of spleen CD3⁺ CD4⁺ T lymphocytes (p < 0.05) when compared with the normal group. However, there was no significant difference in the percentage of spleen CD3⁺ CD8⁺ T lymphocytes among all experimental groups. The finding suggested that an aqueous extract of CO leaves has the ability to reduce blood glucose levels in diabetic rats.